The present invention relates generally to isolated and purified lipoxygenase proteins and nucleic acids. More particularly, the present invention relates to an isolated and purified second type of human 15S-lipoxygenase and an isolated and purified nucleic acid encoding the same, and to an isolated and purified nucleic acid encoding a mouse 8S-lipoxygenase.
The lipoxygenases are a structurally related family of non-heme iron dioxygenases that function in the production of fatty acid hydroperoxides. Three lipoxygenases have been identified and cloned in humans. Funk, C. D. (1993) Prog. Nuc. Acid Res. Mol. Biol. 45:67-98; Matsumoto et al. (1988) Proc. Natl. Acad. Sci. USA 85: 26-30; Dixon et al. (1988) Proc. Natl. Acad. Sci. USA 85: 416-420; Funk et al. (1990) Proc. Natl. Acad. Sci. USA 87: 5638-5642; Izumi et al. (1990) Proc. Natl. Acad. Sci. USA 87:7477-7481; Yoshimoto et al. (1990) Biochem. Biophys. Res. Comm. 172:1230-1235; Sigal et al. (1988) Biochem. Biophys. Res. Comm. 157:457-464). They oxygenate arachidonic acid in different positions along the carbon chain and form the corresponding 5S-, 12S- or 15S-hydroperoxides (hydroperoxy-eicosatetraenoic acids, HPETEs). The three enzymes are known mainly from the blood cell types in which they are strongly expressedxe2x80x94the 5S-lipoxygenase of leukocytes, the 12S-lipoxygenase of platelets, and the 15S-lipoxygenase of reticulocytes, eosinophils and macrophages. While these are the most widely recognized cellular sources, selective expression is well documented in other tissues. For example, both the 12S- and 15S-lipoxygenases are detected in skin. Nugteren et al. (1987) Biochim. Biophys. Acta 921:135-141; Henneicke-von Zepelin et al. (1991) J. Invest. Dermatol. 97:291-297; Takahashi et al. (1993) J. Biol. Chem. 268:16443-16448;Hussain et al. (1994) Amer. J. Physiol. 266:C243-C253.
Potentially, the three cloned lipoxygenases could account for all enzymatic synthesis of arachidonate hydroperoxides in humans, but there are reasons to consider that other lipoxygenases may exist. For example, in the mouse there are five known lipoxygenases, three that correspond to the known human enzymes, Chen et al. (1994) J. Biol. Chem. 269:13979-13987; Chen et al. (1995) J. Biol. Chem. 270:17993-17999 and two others, Furstenberger et al. (1991) J. Biol. Chem. 266:15738-15745; Funk et al. (1996) J. Biol. Chem. 271:23338-23344.
Three of the five distinct mouse lipoxygenase enzymes are best known for their occurrence in different types of blood cells. In common with other mammals, a 5S-lipoxygenase is present in leukocytes and is responsible for synthesis of the pro-inflammatory mediators, the leukotrienes. Chen et al. (1995) J. Biol. Chem. 270:17993-17999; Chen et al. (1994) Nature 372:179-182. A 12S-lipoxygenase is found in platelets and several other tissues including skin. Nugteren et al. (1987) Biochim. Biophys. Acta 921:135-141; Chen et al. (1994) J. Biol. Chem. 269:13979-13987; Sun et al. (1996) J. Biol. Chem. 271:24055-24062.
A second type of 12S-lipoxygenase which is closely related in sequence to the human and rabbit xe2x80x9creticulocyte-typexe2x80x9d of 15S-lipoxygenases occurs in certain macrophages. Sun et al. (1996) J. Biol. Chem. 271, 24055-24062. The fourth mouse lipoxygenase to be characterized is another enzyme to have 12S-lipoxygenase activity; it was cloned recently from mouse skin and has been classified as an epidermal 12S-lipoxygenase. van Dijk et al. (1995) Biochim. Biophys. Acta 1259:4-8; Funk et al. (1996) J. Biol. Chem. 271:23338-23344. All four of these murine lipoxygenases enzymes have been characterized at the cDNA and genomic levels.
The fifth known mouse lipoxygenase was described originally in 1986 by Furstenberger, Marks and colleagues as an enzyme in skin forming 8-HETE and inducible by phorbol ester treatment. Gschwendt et al. (1986) Carcinogenesis 7:449-455. It was shown subsequently that this enzyme forms the 8S enantiomer (Hughes et al. (1991) Biochim. Biophys. Acta 1081:347-354) and isolation of the corresponding hydroperoxide confirmed identification of the enzyme as a lipoxygenase. Fxc3xcrstenberger et al. (1991) J. Biol. Chem. 266:15738-15745. Mouse skin is the only reported site of synthesis of 8S-HETE in animal tissues, and there is no indication from the literature pointing to a potential homologue of the mouse 8S-lipoxygenase in other mammals. Additionally, no nucleic acid, particularly a cDNA, which encodes this lipoxygenase has been characterized.
Despite the description in the art of the enzymes presented above, along with the catalytic activities covered by these enzymes, there remains an open question whether a lipoxygenase rather than a cytochrome P450 might account for the synthesis of 12R-hydroxy arachidonic acid (12R-HETE), Hammarstrxc3x6m et al. (1975) Proc. Natl. Acad. Sci. USA 72:5130-5134; Woollard, P. M. (1986) Biochem. Biophys. Res. Commun. 136(1):169-175; Baer et al. (1991) J. Lipid Research 32:341-347; Holtzman et al. (1989) J. Clin. Invest. 84:1446-1453; Brash et al. (1996) J. Biol. Chem. 271:20549-20557, a prominent arachidonate metabolite in the skin disease of psoriasis and other proliferative dermatoses (Hammarstrxc3x6m et al. (1975) Proc. Natl. Acad. Sci. USA 72:5130-5134; Baer et al. (1991) J. Lipid Research 32:341-347; Baer et al. (1995) J. Invest. Dermatol. 104:251-255).
Therefore, what is needed, then, is further characterization of lipoxygenase enzymes in vertebrates, particularly in mammals, and more particularly in humans. A novel isolated and purified lipoxygenase and a nucleic acid encoding the same would have broad utility to due its role in arachidonic acid metabolism, a critical metabolic pathway.
A key aspect of this invention pertains to the discovery of a novel 15S-lipoxygenase (15-Lox-2) protein and nucleic acid encoding the 15-Lox-2 protein. Preferred nucleic acid and amino acid sequences for 15-Lox-2 are described in SEQ ID NO:1 and SEQ ID NO:2.
It is another aspect of this invention that the novel 15-Lox-2 protein acts in the metabolism of arachidonic acid to 15S-Hydro(pero)xyeicosatetraenoic acid.
Another key aspect of this invention is isolation and purification of a nucleic acid encoding mouse 8S-lipoxygenase (8-Lox) A preferred embodiment of this nucleic acid is described in SEQ ID NO:3.
Thus, in one aspect, the present invention provides an isolated and purified polynucleotide that encodes a lipoxygenase polypeptide wherein the lipoxygenase polypeptide includes an iron ligand comprising a serine. Preferably, the lipoxygenase polypeptide reacts with arachidonic acid. In a preferred embodiment, a polynucleotide of the present invention is a DNA molecule from a vertebrate species. A preferred vertebrate is a mammal. A preferred mammal is a human. More preferably, a polynucleotide of the present invention encodes polypeptides designated 15-Lox-2 and 8-Lox. Even more preferred, a polynucleotide of the present invention encodes a polypeptide comprising the amino acid residue sequence of SEQ ID NO:2 or SEQ ID NO:4. Most preferably, an isolated and purified polynucleotide of the invention comprises the nucleotide base sequences of SEQ ID NO:1 or SEQ ID NO:3 or their homologues from other vertebrate species.
Yet another aspect of the present invention contemplates an isolated and purified polynucleotide comprising a base sequence that is identical or complementary to a segment of at least 10 contiguous bases of SEQ ID NO:1 wherein the polynucleotide hybridizes to a polynucleotide that encodes a lipoxygenase polypeptide wherein the lipoxygenase polypeptide includes an iron ligand comprising a serine. Preferably, the lipoxygenase polypeptide reacts with arachidonic acid. Preferably, the isolated and purified polynucleotide comprises a base sequence that is identical or complementary to a segment of at least 25 to 70 contiguous bases of SEQ ID NO:1. For example, a polynucleotide of the invention can comprise a segment of bases identical or complementary to 40 or 55 contiguous bases of the disclosed nucleotide sequences.
In another embodiment, the present invention contemplates an isolated and purified lipoxygenase polypeptide wherein the lipoxygenase polypeptide includes an iron ligand comprising a serine. Preferably, the lipoxygenase polypeptide reacts with arachidonic acid. More preferably, a polypeptide of the invention is a recombinant polypeptide. Even more preferably, a polypeptide of the present invention is 15-Lox-2. Even more preferably, a polypeptide of the present invention comprises the amino acid residue sequence of SEQ ID NO:2.
In an alternative embodiment, the present invention provides an expression vector comprising a polynucleotide that encodes a lipoxygenase polypeptide that includes an iron ligand comprising a serine. Preferably, the lipoxygenase polypeptide reacts with arachidonic acid. Also preferably, an expression vector of the present invention comprises a polynucleotide that encodes 15-Lox-2 or 8-Lox. More preferably, an expression vector of the present invention comprises a polynucleotide that encodes a polypeptide comprising the amino acid residue sequence of SEQ ID NO:2 or SEQ ID NO:4. More preferably, an expression vector of the present invention comprises a polynucleotide comprising the nucleotide base sequence of SEQ ID NO:1 or SEQ ID NO:3. Even more preferably, an expression vector of the invention comprises a polynucleotide operatively linked to an enhancer-promoter. More preferably still, an expression vector of the invention comprises a polynucleotide operatively linked to a prokaryotic promoter. Alternatively, an expression vector of the present invention comprises a polynucleotide operatively linked to an enhancer-promoter that is a eukaryotic promoter, and the expression vector further comprises a polyadenylation signal that is positioned 3xe2x80x2 of the carboxy-terminal amino acid and within a transcriptional unit of the encoded polypeptide.
In yet another embodiment, the present invention provides a recombinant host cell transfected with a polynucleotide that encodes a lipoxygenase polypeptide which includes an iron ligand comprising a serine. Preferably, the lipoxygenase polypeptide reacts with arachidonic acid. SEQ ID NO:1; SEQ ID NO: 2 SEQ ID NO:3; and SEQ ID NO: 4 set forth nucleotide and amino acid sequences from the exemplary vertebrates human and mouse. Also contemplated by the present invention are homologous or biologically equivalent polynucleotides and lipoxygenase polypeptides found in other vertebrates. Preferably, a recombinant host cell of the present invention is transfected with the polynucleotide that encodes 15-Lox-2 or 8-Lox. More preferably, a recombinant host cell of the present invention is transfected with the polynucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3. Even more preferably, a host cell of the invention is a eukaryotic host cell. Still more preferably, a recombinant host cell of the present invention is a vertebrate cell. Preferably, a recombinant host cell of the invention is a mammalian cell.
In another aspect, a recombinant host cell of the present invention is a prokaryotic host cell. Preferably, a recombinant host cell of the invention is a bacterial cell, preferably a strain of Escherichia coli. More preferably, a recombinant host cell comprises a polynucleotide under the transcriptional control of regulatory signals functional in the recombinant host cell, wherein the regulatory signals appropriately control expression of a lipoxygenase polypeptide that metabolizes arachidonic acid in a manner to enable all necessary transcriptional and post-transcriptional modification.
In yet another embodiment, the present invention contemplates a process of preparing a lipoxygenase polypeptide comprising transfecting a cell with polynucleotide that encodes a lipoxygenase polypeptide which includes an iron ligand comprising a serine, to produce a transformed host cell; and maintaining the transformed host cell under biological conditions sufficient for expression of the polypeptide. Preferably, the lipoxygenase polypeptide that is produced reacts with arachidonic acid. More preferably, the transformed host cell is a eukaryotic cell. More preferably still, the eukaryotic cell is a vertebrate cell. Alternatively, the host cell is a prokaryotic cell. More preferably, the prokaryotic cell is a bacterial cell of the DH5xcex1 strain of Escherichia coli. Even more preferably, a polynucleotide transfected into the transformed cell comprises the nucleotide base sequence of SEQ ID NO:1 or SEQ ID NO:3. SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; and SEQ ID NO:4 set forth nucleotide and amino acid sequences for the exemplary vertebrates human and mouse. Also contemplated by the present invention are homologues or biologically equivalent lipoxygenase polynucleotides and polypeptides found in other vertebrates.
In still another embodiment, the present invention provides an antibody immunoreactive with a lipoxygenase polypeptide which includes an iron ligand comprising a serine. Preferably, the lipoxygenase polypeptide reacts with arachidonic acid. SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; and SEQ ID NO:4 set forth nucleotide and amino acid sequences from the exemplary vertebrates human and mouse. Also contemplated by the present invention are antibodies immunoreactive with homologues or biologically equivalent lipoxygenase polynucleotides and polypeptides found in other vertebrates. Preferably, an antibody of the invention is a monoclonal antibody. More preferably, the lipoxygenase polypeptide comprises 15-Lox-2 or 8-Lox. Even more preferably, a polypeptide comprises the amino acid residue sequence of SEQ ID NO:2 or SEQ ID NO:4.
In another aspect, the present invention contemplates a process of producing an antibody immunoreactive with a lipoxygenase polypeptide which includes an iron ligand comprising a serine, the process comprising the steps of (a) transfecting a recombinant host cell with a polynucleotide that encodes a lipoxygenase polypeptide which includes an iron ligand comprising a serine; (b) culturing the host cell under conditions sufficient for expression of the polypeptide; (c) recovering the polypeptide; and (d) preparing the antibody to the polypeptide. Preferably, the lipoxygenase polypeptide reacts with arachidonic acid. SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; and SEQ ID NO:4 set forth nucleotide and amino acid sequences from the exemplary vertebrates mouse and human. Preferably, the host cell is transfected with the polynucleotide of SEQ ID NO:1 or SEQ ID NO:3. Even more preferably, the present invention provides an antibody prepared according to the process described above. Also contemplated by the present invention is the use of homologues or biologically equivalent polynucleotides and polypeptides found in other vertebrates to produce antibodies.
Alternatively, the present invention provides a process of detecting a lipoxygenase polypeptide that metabolizes arachidonic acid, wherein the process comprises immunoreacting the polypeptide with an antibody prepared according to the process described above to form an antibody-polypeptide conjugate, and detecting the conjugate.
In yet another embodiment, the present invention contemplates a process of detecting a messenger RNA transcript that encodes a lipoxygenase polypeptide which includes an iron ligand comprising a serine, wherein the process comprises hybridizing the messenger RNA transcript with a polynucleotide sequence that encodes that polypeptide to form a duplex; and detecting the duplex. Alternatively, the present invention provides a process of detecting a DNA molecule that encodes a lipoxygenase polypeptide, wherein the process comprises hybridizing DNA molecules with a polynucleotide that encodes a lipoxygenase polypeptide which includes an iron ligand comprising a serine to form a duplex; and detecting the duplex. For both such processes, it is preferred that the detected lipoxygenase polypeptide is capable of reacting with arachidonic acid.
In another aspect, the present invention contemplates a diagnostic assay kit for detecting the presence of a lipoxygenase polypeptide in a biological sample, where the kit comprises a first container containing a first antibody capable of immunoreacting with a lipoxygenase polypeptide which includes an iron ligand comprising a serine, with the first antibody present in an amount sufficient to perform at least one assay. Preferably, an assay kit of the invention further comprises a second container containing a second antibody that immunoreacts with the first antibody. More preferably, the antibodies used in an assay kit of the present invention are monoclonal antibodies. Even more preferably, the first antibody is affixed to a solid support. More preferably still, the first and second antibodies comprise an indicator, and, preferably, the indicator is a radioactive label or an enzyme.
In an alternative aspect, the present invention provides a diagnostic assay kit for detecting the presence, in biological samples, of a lipoxygenase polypeptide, the kits comprising a first container that contains a second polynucleotide identical or complementary to a segment of at least 10 contiguous nucleotide bases of a polynucleotide that encodes a lipoxygenase polypeptide which includes an iron ligand comprising a serine. Preferably, the polynucleotide encodes a lipoxygenase polypeptide capable of reacting with arachidonic acid. More preferably, the polynucleotide encodes 15-Lox-2 or 8-Lox.
In another embodiment, the present invention contemplates a diagnostic assay kit for detecting the presence, in a biological sample, of an antibody immunoreactive with a lipoxygenase polypeptide, the kit comprising a first container containing a lipoxygenase polypeptide which includes an iron ligand comprising a serine that immunoreacts with the antibody, with the polypeptide present in an amount sufficient to perform at least one assay. Preferably, the lipoxygenase polypeptide is capable of reacting with arachidonic acid. More preferably, the polypeptide comprises 15-Lox-2 or 8-Lox.
The foregoing aspects and embodiments have broad utility given the biological significance of the arachidonic acid pathway, as is known in the art. By way of example, the foregoing aspects and embodiments are useful in the preparation of screening assays and assay kits that are used to identify compounds that affect arachidonic acid metabolism, or that are used to detect the presence of the proteins and nucleic acids of this invention in biological samples. Additionally, it is well known that isolated and purified polypeptides have utility as feed additives for livestock and further polynucleotides encoding the polypeptides are thus useful in producing the polypeptides.
Following long-standing patent law convention, the terms xe2x80x9caxe2x80x9d and xe2x80x9canxe2x80x9d mean xe2x80x9cone or morexe2x80x9d when used in this application, including the claims.
Some of the aspects and objects of the invention having been stated hereinabove, other aspects and objects will become evident as the description proceeds, when taken in connection with the accompanying drawings as best described hereinbelow.